Gene Mapping And Candidate Gene Analysis Of White Petal Dominant Gene In Brassica Napus L.

Variations in flower color is from a wide variety of sources in Brassica napus L..Although the most common petal color is yellow,there are still a few of different colors,such as white,pink,orange,etc.The flower color has advantages trait of easy observation and genetic stability,and play a role in detecting the natural outcrossing rate and hybrid purity of varieties in Brassica napus L.Meanwhile,the flower color has high ornamental value and can be used as tourism resources to promote regional economic development.Therefore,the research on the candidate interval of white petal genes based on next generation sequencing（NGS）and the analysis of candidate genes can lay the foundation of theory and provide a new approach to flower color molecular breeding.In this study,we carried out genetic analysis of white petal color with F₂ mapping population,and adopt the approach of plant breeding in combination with QTL-seq,mutation mapping analysis pipeline for pooled RNA-seq（MMAPPR）,transcriptome and alternative splicing,to locate candidate interval and linkage markers of the white petal gene,to find out differential expression of candidate genes,to screen candidate genes of white petal with reference sequence and annotated documents of Darmor-bzh.The results are as follows:1.Genetic analysis of petal colorAn inbred line Y05,which has yellow petal,was crossed with an inbred line W01,which has white petals.The F₁ plants were self-crossed to develop F₂ mapping population including 1437 individuals.The segregation of white petal（1093）and yellow petal（345）among F₂ population fitted the Mendelian segregation ratio of 3:1(χ²=0.7810,χ2_0.05,1=3.84,P>0.05).This indicates that the white petal trait was controlled by a major gene and that white petal was dominant over yellow petal.2.Mapping of white dominant petal geneFor bulked segregant analysis（BSA）,parental and two pools with 30 yellow petal lines and 30 white petal lines of F₂ were constructed by mixing an equal amount of DNA or RNA respectively.30×or 5×depth of genome sequencing was conducted.The results of the candidate interval using QTL-seq that a candidate interval（49-56.5 Mb）exceeding the threshold value was identified for the petal color on chromosome C03 when Darmor-bzh was used as reference genome.While ZS11,Darmor and Tapidor were aligned to sequence data,candidate intervals for white petal were all identified on chromosome C03.Linked region peaks（54-56 Mb）identified by MMAPPR for the petal color was on chromosome C03 of Darmor-bzh.In candidate interval,the simple sequence repeat（SSR）primers were designed by MISA and Prime3 for repeated sequence identification.The insertion-deletion（InDel）sites can be visualized in candidate interval by integrative genomics viewer（IGV）.Based on these Indel variations,we used Vector and Blast to design InDel primers.Using 54polymorphic SSR and InDel markers,white petal gene was primary mapped between markers InDel-2 and SSR-139 by BSA,the genetic distance respectively were 0.29 c M and0.58 c M,the interval on reference genome is about 254 kb.Nineteen annotated genes were found on the 254 kb target region of the Darmor-bzh reference genome V4.1,and the 19genes were analyzed with the homologous Arabidopsis gene sequence to identify the possible candidate gene.3.Analysis of transcriptome and alternative splicingTwo biological repeat transcriptome analysis were performed on the progeny pool of yellow petal and progeny pool of white petal in F₂ segregating population and 15differentially expressed genes（DEGs）at 0.05 level were identified.Among them,Bna C03g63070D was between markers Indel-2 and SSR-139.In the KEGG enrichment analysis,the Bna A08g09110D was significantly enriched in the carotenoid biosynthesis pathway,and Bna A05g04030D was significantly enriched in the phenylpropane biosynthesis pathway.Although Bna C03g63070D had no functional annotation,it was clustered together with Bna A05g04030D in the cluster analysis of differentially expressed genes,and it was found that Bna C03g63070D and Bna A08g09110D had homologous collinear according to analysis of gene sequence.So it is speculated that Bna C03g63070D may play a role in anthocyanin synthesis.The alternative spilcing results showed that Bna C03g63070D had no alternative splicing events in white petal bud stage and yellow petal full-bloom stage,but intron retention and the 3’-end alternative spilcing events occurred in yellow petal bud stage,and the intron retention events occurred in white petal full-bloom stage.The results of quantitative real-time PCR（qRT-PCR）confirmed that the expression level of Bna C03g63070D in white flower petals was significantly higher than that in yellow flower petals.Combined with the analysis of the initial localization region of white flower dominant gene,differentially expressed genes and alternative splicing,it was suggested that Bna C03g63070D might be involved in pigment synthesis in petals.